The overall objective of this competitive renewal is to identify the key modulators of post-traumatic osteoarthritis (PTOA) using a novel large translational model of ACL injury and bio-enhanced ACL repair. In the last funding cycle we demonstrated with our experimental model that accelerated PTOA occurs following ACL injury and even after ACL reconstruction, the current standard of care in which the injured ligament is replaced by a tendon graft. We determined that our novel technique of bio-enhanced ACL repair, where the injured ligament is repaired using a bio-engineered scaffold to stimulate healing, not only effectively heals the ligament, but also prevents the onset of PTOA after ACL injury. The mechanisms behind this therapeutic effect on cartilage are unknown and the focus of the proposal. With the translational experimental model developed in the last funding cycle, we are now poised to use a differential systems biology approach to compare subjects that do not develop PTOA (i.e., those treated with bio-enhanced ACL repair) with those that do (i.e., those treated with conventional ACL reconstruction) to determine key biological, structural and biomechanical pathways that are different in knees which are developing PTOA from those that are not. The specific aims of the study are: 1) to identify differences in cytokine, extracellular matrix (ECM)-associated protein profiles and asymmetries in gait in the joints of individuals undergoing ACL transection followed by bio-enhanced ACL repair compared to individuals undergoing ACL transection followed by conventional ACL reconstruction at 1, 4, 12, 26 and 52 weeks after injury, and 2) to define the source of cytokine and ECM-related protein expression within the synovial joint at 1, 4, and 52 weeks after injury. Both aims will take advantage of our well- characterized experimental model of ACL transection, ACL reconstruction and bio-enhanced ACL repair. The primary outcome measures for Aim 1 (longitudinal assessments) are the concentrations of the cytokines, MMPs, and collagen and aggrecan fragments in synovial fluid, and the ratio of the peak ground reaction force during stance phase of gait. The primary outcome measures for Aim 2 (cross-sectional assessments) are the gene expression assays of cytokines in the synovium, ligament and articular cartilage and macroscopic (modified outerbridge score) and histological assessments of cartilage health. At the completion of this study we will better understand the chondroprotective effects of the bio-enhanced ACL repair, establish upstream targets for therapeutic intervention for other traumatic joint injuries, and be poised to transfer the bio-enhanced repair technology for clinical use.